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VET 204: Veterinary Clinical Laboratory
Techniques
Leukocytes Introduction:Leukocytes (white blood cells) are responsible for protecting the body against foreign invaders, such as bacteria, viruses, fungi and parasites. Unlike erythrocytes, leukocytes are nucleated and capable of independent movement. Their life spans range from a few hours to several years, depending upon type of leukocyte. White blood cells (WBCs) are produced in several locations. The stem cells are located in the bone marrow, and most leukocytes are produced there. As the young animal grows, however, lymphocyte stem cells colonize lymphoid tissues throughout the animal's body, and production and specialization occurs at these sites. In general, leukocytes function in the tissues, moving out of the blood vessels and into the cells in the region where their immune activities are needed. Most of the WBCs complete their lives and die at the site of activity, but lymphocytes are capable of recirculating, by moving into the lymphatic vessels and then entering back into general circulation. The primary function of leukocytes is to provide protection to their animal. Skin, mucous membranes and digestive activities provide a first line of defense. If pathogens, such as bacteria, fungi, parasites and viruses, are able to pass the initial defenses, it is the role of the WBC to provide protection. Most of the leukocytes (neutrophil, basophil, eosinophil and monocyte) provide nonspecific immunity. Their action is innate- it is inherited by the animal and is effective against a broad range of pathogens. Part of the innate response is inflammation, a wide-ranging physiologic response that "marshals" the body to resist the effects of infection and trauma. The general innate responses are:
Lymphocytes are responsible for adaptive immunity- an
immune response that is developed by the animal against specific
antigens after exposure to those antigens. Evaluation of Leukocytes:
Total Leukocyte Count: The most accurate method to enumerate leukocytes is to use an automatic blood cell counter. There are a variety of machines available and the error in results from using an automated count is approximately one to two percent. Both impedance analyzers and manual counting methods require the lysis of erythrocytes prior to counting white blood cells (only nonnucleated erythrocytes are lyzed). Because of the expense of automated equipment, hospitals may use a manual count to determine the WBC count. This method is less accurate (with an error of up to 10%), but it is also less expensive. The manual method generally uses a commercially supplied product, the Unopette®, which consists of a pipet and reservoir containing a premeasured volume of diluent. The cells are counted on a hemacytometer, special microscope slide with an etched counting chamber. In western New York, the most practices use Unopette® 5855. All nine supersquares on the hemacytometer grid are counted, ten percent of the total is added to the count and the sum is multiplied by the correction factor of 100. Nonmammals have nucleated erythrocytes that don't
lyse. Some automated systems are capable of
counting avian,
reptile and other nonmammal blood cells. The most common
method used to enumerate WBCs in these species is to use Unopette®
5877, which stains eosinophilic cells, and then correct the count
using a differential leukocyte count. Some practices use a special diluent, Natt
and Herrick's solution, that is specifically designed for counting
avian leukocytes.
Leukopenia is a decrease in the number of total leukocytes. This also may be caused by different factors, including:
The "differential" is used to determine the number of each cell type present in a specimen. There are two types of differentials: relative and absolute. The relative differential is determined from a stained smear, on which at least 100 cells are counted and classified. (In animals with a leukocytosis, more than 100 cells should be counted to obtain a representative sample.)
Differentials should always be recorded and interpreted as absolute rather than relative values. If, for example, a dog has a leukopenia with a the total leukocyte count is 5,000/ml, it might appear that the cell counts were normal if you looked at the relative differential and 65% of the cells were segmented neutrophils. In fact, the reference range for segmentors in the dog is 6,9000 - 8,900/ml...and this dog's absolute segmented neutrophils is only 3,250/ml, well below the reference range. If the relative differential for lymphocytes is 65% (extremely high) in the same dog, the absolute number of 3,250/ml is "normal" compared to the dog's reference range for lymphocytes of 1,400 - 3,400/ml. Leukocyte Kinetics and Morphology: Neutrophils (Segs, Segmentors, Polymorphonuclear cells or PMNs) Neutrophils are classed as "granulocytes"--there are granules present in the cytoplasm, although these granules do not stain. These leukocytes are responsible for short-term phagocytosis during the initial stage of an acute bacterial infection. Neutrophil Kinetics: Like the erythrocyte, the neutrophil develops from a pluripotent stem cell in the bone marrow. The complete neutrophil maturation sequence is:
Neutrophil production and maturation occurs in the bone marrow (the intramedullary phase) and in circulation (the intravascular phase). In addition, there is a tissue phase where the neutrophils perform their phagocytic responsibilities. The intramedullary and intravascular phases are divided into "pools"- these are imaginary areas that are designated as sites for specific activities. In the bone marrow, for example, there are proliferating, maturation and reserve pools. These pools don't have specific areas within the bone marrow--proliferating occurs throughout the bone marrow, but this convention of naming pools helps in visualizing what is occurring. The proliferating pool is the area where mitotic cell division occurs. This includes the stem cell through the myelocyte. These cells actively dividing, but are not capable of phagocytosis. Cells in the maturation pool are no longer capable of division; they are undergoing internal changes that allows them to develop from metamyelocytes to band neutrophils. There is also a reserve pool in the bone marrow. This pool consists of mature cells that are not yet in circulation. Only segmentors are found in this pool. Cells are released from the bone marrow into circulation in an age-related order. Segmented neutrophils from the reserve are the first cells released in response to increased need for phagocytosis. When these are exhausted, the cells from the maturation pool are released, first the band neutrophils and then, if demand still exists, the metamyelocytes. The most mature neutrophils are the most effective phagocytizers, so the age-related release provides the most effective cells first. There are two intravascular pools: the circulating and the marginated pools. The circulating pool are the neutrophils found in the main stream of a blood vessel and these are the cells that are collected during venipuncture and then counted. The rest of the neutrophils are found in the marginated pool. These cells are skimming along the endothelial surface of the blood vessels, especially in the capillaries. They will be the first neutrophils that move from the intravascular phase into the tissue phase. Movement of neutrophils through the intercellular junctions of the endothelium into the tissues is called diapedesis. Most neutrophils only circulate through the blood vessels for a few hours before they enter the tissues. Once they are in the tissues, the neutrophils have very short life spans--generally only 1 - 2 days before their bacterial fighting reserves are exhausted and they are destroyed in the monocyte-macrophage system. Neutrophil Function: Neutrophilic metamyelocytes, bands and segmentors are all capable of phagocytosis. They are the first leukocyte line of defense and destroy bacteria during the initial stage of acute infections. Movement of neutrophils toward the site of infection begins with the "migration cascade," a series of steps that bring the neutrophil from the blood vessels to the site where it is needed. Cells damaged by bacteria release chemicals into the surrounding tissues. These chemicals attract neutrophils by a process called chemotaxis. The neutrophils congregate in the blood vessels when the concentration of the chemicals is strongest. The cells marginate and then pass through the endothelial junctions by diapedesis and migrate through the tissues to the location of the bacteria. At the infection site, the neutrophils recognize the bacteria and attach to them. This attachment is enhance by opsonization, which occurs when bacteria have been coated by inflammatory proteins such as complement. The neutrophils destroy the bacteria by engulfing them and exposing them to lytic enzymes and toxic oxygen products. Neutrophilic Variations: The heterophil is sometimes called a "pseudoeosinophil." It is a neutrophil that contains pink-staining granules, but is morphologically and functionally distinct from eosinophils. Heterophils occur in birds, rodents, rabbits, elephants and other species. Care must be taken when performing a differential on these animals so that eosinophils are differentiated from the heterophils. An increase in neutrophils is called neutrophilia and this is the primary cause of leukocytosis in small animals. It is important to determine the cause of a neutrophilia.
A decrease in neutrophils is called neutropenia.
A shift to the left occurs when immature neutrophils are present (i.e. high numbers of bands and possibly metamyelocytes). This commonly indicates that infection is present, as less mature neutrophils are released from the bone marrow. These cells may be toxic, with morphologic changes present. A neutrophil with a shift to the right is hypersegmented--there are at least 5 lobes to the nucleus. This is a segmentor that has been retained in circulation for an extended period of time. This may be a result of normal aging, where the occasional hypersegmented cell has not yet marginated and moved into the tissues. Increased numbers of hypersegmented cells are associated with stress or administration of corticosteroids. The presence of toxic neutrophils indicates toxemia--the presence of toxins within the blood stream that causes morphologic damage to the cells. Less than 5% toxic neutrophils is considered insignificant.
Pathologic inclusions from may accompany some infections. Distemper inclusion bodies are irregularly-shaped magenta inclusions (similar, though paler, inclusions also occur in the erythrocytes of animals with distemper). Ehrlichia-infected neutrophils contain small grayish mulberry-like inclusions (morulae).
Eosinophil Kinetics: Like neutrophils, eosinophils are granulocytes and their production and maturation is similar to that of the neutrophil. Eosinophil Functions: The primary function of eosinophils is to help control inflammation. They do this by:
The eosinophil also phagocytizes pathogens. It is most effective against migrating nematode larvae, often coating the surface of the larvae and destroying its cuticle. Eosinophilic Variations: An increase in the number of eosinophils is called
eosinophilia. It is frequently seen in animals with
allergies, especially food allergies and feline asthma. Parasitic
infections due to fleas, canine heartworm and lungworm are also
accompanied by eosinophilia. Eosinophilic granulomas also results
in eosinophilia. Basophils are almost never seen on a blood smear. Basophil Kinetics: Like neutrophils, basophils are granulocytes and their production and maturation is similar to that of the neutrophil. Basophil Functions: Basophils are also involved with inflammation, but basophils help initiate inflammation. They release chemical mediators such as heparin, histamine and serotonin. Basophils are capable of limited phagocytosis. Mast cells are the tissue equivalent of basophils, and may be seen in impression smears of affected tissues. Basophilic Variations: Basophilia is rare, but may be seen in heartworm disease (along with an eosinophilia), chronic respiratory infections and mast cell tumors. Basopenia is not detectable, because basophils are rarely seen in
circulation. Monocytes are agranulocytes- there are no granules seen in the
cytoplasm. Approximately 5 - 10% of the leukocytes in circulation
are monocytes. Lymphocytes |
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Reading
assignment: Rebar- white blood cell chapters (5 - 9)